The goal of this proposal is to apply innovative technologies to advance novel sets of inhibitors against the rhamnnose and galactose cell wall biosynthesis enzymes of M. tuberculosis. Three groups of inhibitor "hits" discovered in the parent grant will be developed into non-toxic, lead compounds that inhibit both their target enzymes and bacterial growth at low concentrations. Hit compounds will progress through a technologically advanced drug development program that includes: (1) structure and diversity guided compound library design; (2) X-ray crystal structure analysis of inhibitor/enzyme complexes; (3) high-throughput parallel combinatorial synthesis. This response to PAS-02-031 is designed to capitalize on the advancements made under the parent grant, AI-33706, in which four enzymes, Rml B-D and GIf, have been shown to be essential for M. tuberculosis growth. Also under the parent grant, inhibition assays for each enzyme were developed, "drug-like" structurally diverse libraries were screened (using additional support from AI-46393), and three classes of active compounds were identified. In this proposal, a structure guided drug development cycle will be implemented to optimize each class of active compounds. Initially, computer modeling and the known X-ray structures of the four enzymes will be used to determine the interactions between identified inhibitors and the active-site ligands of their target enzyme. Co-crystal structures of selected hits will be determined by X-ray crystallography and subsequent in silico libraries will be designed based on each of the three scaffolds. The libraries will be filtered for compounds that have low toxicity, good pharmacokinetics, tight binding to the active site and substantial chemical diversity. These optimized libraries will then be synthesized via high-throughput, parallel synthesis and tested for enzyme inhibition, anti-mycobacterial activity against M. tuberculosis, and selective toxicity in human cell lines. This cycle will then be repeated until highly active, non-toxic leads are produced. The innovative drug development program in this proposal will complement research on the parent grant and significantly progress this research forward, towards the ultimate goal of developing more effective drugs for the treatment of tuberculosis.